Subtilisin Carlsberg

P0 Gallus domesticus chickens were dissected as described previously [28] (link), [29] (link); the tectorial membrane was removed with subtilisin Carlsberg (Sigma Type XXIV) protease (50 µg/ml for 15 min). Extracellular solution was used at room temperature for all dissecting, loading, and imaging steps and contained the following (in mM): 87 NaCl, 0.5 KCl, 0.5 CaCl₂, 1.25 NaH₂PO₄, 2 ascorbate, 2 creatine, 6 Na-pyruvate, 75 sucrose, 25 D-glucose, 10 HEPES (pH 7.4, 310–320 mOsm). Fluo-4 AM dye was prepared as described in Figure 1, using the following reagents: Fluo-4 AM (Invitrogen, F14201), DMSO (Invitrogen, C6667), Pluronic F-127, 20% solution in DMSO (Invitrogen P3000MP). Bringing all dye reagents to room temperature before beginning was essential; moreover, all preparation and loading steps were carried out at room temperature in foil-wrapped tubes to protect dye fluorescence. To normalize for dye loading in fluid-jet experiments, the cell-permeable inert dye CellTracker Red CMTPX (CT-Red; Invitrogen C34552) was added to the organs for 15 min following Fluo4-AM loading. There was no obvious difference in the Fluo-4 signal between tall and short hair cells, and both cell types were analyzed in the group data.

The simultaneous effect of two factors, temperature (T) and pH, on the enzymatic hydrolysis of S. canicula muscle by-products was evaluated by a rotatable second order design [35 ]. The values of the variables tested for each protease and the procedure of codification-decodification of the variables is summarized in Table S1. The commercial proteases were Alcalase 2.4 L (2.4 Anson Unit/g, AU/g), Esperase 8 L (8 KNovo Protease Unit/g, KNPU/g) and Protamex (Novozymes, Nordisk, Bagsvaerd, Denmark), and the concentration employed was 1% (v/w of muscle) in all cases. The rest of the experimental conditions were maintained constant: ratio solid:liquid (1:5) and 200 rpm of agitation. The kinetics of hydrolysis were performed in a controlled pH-Stat system with a 100 mL glass-reactor and extended up to 6 h. At the end of hydrolysis, the samples were heated at 90 °C for 15 min to inactivate the proteases and were stored at −20 °C until analysis.
Orthogonal least-squares calculation on factorial design data were used to obtain the empirical equations describing the different dependent variables (hydrolysis kinetic parameters and bioactivities) assessed (Y) in function of the independent variables (T and pH):
$Y=b_0+b_{1}T+b_{2}pH+b_{12}TpH+b_{11}{T}^2+b_{22}p{H}^2,$
where Y represents the parameters to be modelled; b₀ is the constant coefficient, b₁ and b₂ are the coefficient of linear effects, b₁₂ is the coefficient of interaction effect among pH and T, and b₁₁ and b₂₂ are the coefficients of quadratic effects. The Student t-test (α = 0.05) was employed to determine the statistical significance of the coefficients. The goodness-of-fit was established as the determination coefficient (R²) and the model consistency by the Fisher F-test (α = 0.05) using the following mean squares ratios:
the model is acceptable whenF1 = Model/Total errorF1≥FdennumF2 = (Model + Lack of fitting)/ModelF2≤FdennumF3 = Total error/Experimental errorF3≤FdennumF4 = Lack of fitting/Experimental errorF4≤Fdennum $F_{den}^{num}$ are the theoretical values to α = 0.05 with the corresponding degrees of freedom for numerator (num) and denominator (den). The equation is acceptable when F1 and F2 are validated. F3 and F4 were additionally calculated to improve the degree of robustness and consistency of the empirical equations obtained. A Microsoft Excel spreadsheet was employed for the procedures of numerical fittings, coefficient estimates, and statistical evaluations.

All chemicals were purchased from Merck KGaA (Darmstadt, Germany) with purity >98% unless otherwise noted. A commercial formulation of the ESP Subtilisin Carlsberg (SC) from Bacillus licheniformis, type VIII, (Merck, cat. no. P5380) was included in activity assays.

Immediately after harvest liver, quadriceps muscle, and kidneys were placed in ice-cold STE1 buffer (250 mmol/L sucrose, 5 mmol/L Tris/HCl, 2 mmol/L EGTA, pH 7.4). Tissues were minced and either incubated in 2.5 mL STE2 buffer (STE1 containing [wt/vol] 0.5% BSA, 5 mmol/L MgCl₂, 1 mmol/L ATP, and 2.5 U/mL protease Subtilisin A) for 4 min (quadriceps muscle) or immediately proceeded to homogenization (liver and kidney tissue). All tissues were homogenized using a Teflon pistil in a Potter-Elvejhem homogenizer. Quadriceps muscle homogenates were further diluted with 2.5 mL STE1 buffer containing Complete Mini protease inhibitor cocktail (Roche, Mannheim, Germany), centrifuged at 8,000 g for 10 min, and the resulting pellet was resuspended in 4 mL STE1 buffer. Next, all tissues homogenates were centrifuged at 800 g for 10 min and supernatants were centrifuged at 8,000 g for 10 min. Pellets obtained from mitochondrial isolation were resuspended in 200 µL STE1 buffer each. State III oxygen consumption rates (V_ADP) were determined in 300 µg of mitochondria by using a Clark-type oxygen electrode (Strathkelvin, North Lanarkshire, Scotland) with 20 µmol/L palmitoyl-carnitine (PC)/2 mmol/L malate or 10 mmol/L pyruvate/5 mmol/L malate as substrates as previously described (14 (link)).

Rice protein was obtained through precipitation per the method of Chen et al.^{45 (link)}. In brief, rice protein was enzymatically hydrolyzed with a 2:100 (v/v) enzyme-to-substrate ratio to obtain hydrolysates. Protease (Alcalase, EC 3.4.21.62, serine endo-peptidase, 2.4 AU-A/g; Healthmate, Changhua, Taiwan) was used and the hydrolysis conditions involved setting pH 8.0 at 50 ± 5 °C. After a 4-h reaction period, the protease was inactivated through heating at 85 °C for 15 min. After centrifugation (5000 rpm, 4 °C) for 15 min, the supernatant was lyophilized and stored at − 20 °C before use.